Summary: 1. Development of the first international standard for von Willebrand factor (vWF) concentrates The development of the first international standard for vWF concentrates is a joint effort among CBER/FDA, NIBSC, and the Science and Standardization Committee (SSC) of the International Society on Thrombosis and Haemostasis. The initial characterization of five vWF concentrates was performed at three sites (CBER/FDA, NIBSC, and Laboratoire Francais du Fractionnement et des Biotechnologies). The results of the study were reported to the vWF subcommittee at the SSC annual meeting on June 16, 2000 in Maastricht, The Netherlands. The SSC has accepted the selection of two candidates for final phase of production and calibration. The two candidates were selected based on parallelism of dose-response curves, stability of preparations over accelerated and long term storage (i.e. stability of vWF multimers, antigen, and potency), similar results between different test methodologies (e.g. Ristocetin Cofactor Activity assay versus Collagen Binding assay), the ratio between activity and antigen, and the integrity of vWF multimers. Two large fills were prepared for an international calibration study. Twenty-six laboratories worldwide have participated. The results of the study were presented at the SSC annual meeting on July 6, 2001 in Paris. The SSC accepted the study report with proposed potency assignment. This work was presented to the World Health Organization Expert Committee on Biological Standardization (ECBS/WHO) for final establishment in October 2001 and accepted by ECBS/WHO in November 2001. The report of this work has been accepted by the official journal of ISTH, Thrombosis and Haemostasis. 2. Development of Mega-2, a working standard for factor VIII activity Supplies of Mega-1, the U.S. working standard for coagulation factor VIII are low. The Center for Biologics Evaluation and Research (CBER), FDA has, therefore, initiated efforts to replace the Mega-1 standard with Mega-2. Mega-2 is a plasma-derived factor VIII concentrate. It was selected based on the integrity of the factor VIII molecule, linearity of the dose-response over a wide range of concentrations, minimal inter- and intra-assay variability, stability of factor VIII potency over accelerated and long term storage, and consistent results between different test methodologies (e.g. one-stage APTT assay versus chromogenic substrate assay). The final fill of Mega-2 was performed in May 2000, and 100,000 vials will be available for distribution. During fiscal year 2001, the final phase of development for the Mega 2 standard for coagulation Factor VIII was completed. FDA/CBER collaborated with the European Pharmacopeia/the European Directorate for the Quality of Medicines (EP/EDQM) in organizing an international collaborative effort to calibrate the Mega 2 standard. The standard was calibrated against four (4) established FVIII standards using both the chromogenic substrate and one stage clotting assay methods. In addition a similar material, filled into sealed glass ampoules at the National Institute for Biological Standards and Control (NIBSC) (concentrate N) was calibrated. Thirty eight (38) laboratories from 21 countries participated. Thirty-one (31) labs calibrated the Mega 2 and N standards using the chromogenic substrate method. Twenty-seven (27) laboratories calibrated both standards by the one stage method. Eighteen (18) laboratories performed both methods. The final potencies assigned to the Mega 2 standard were: 8.6 IU/vial by chromogenic substrate method and 11.3 IU/vial by one stage clotting method. The final potencies determined for concentrate N were: 10.7 IU/ampoule chromogenic substrate method and 10.5 ampoule by one stage clotting method. 3. Development of an International Thrombin Standard with an Unified Unit FDA is obliged to provide national standard for thrombin according to the regulation. With the rapid depletion of both the U.S. national standard for thrombin (Lot J) and the international standard for a-thrombin (89/588), CBER and the National Institute for Biological Standards and Control (NIBSC) collaborated on a project to replenish the inventory for a new international standard for thrombin. In addition, this project also aims to eliminate the difference between the U.S. and international units for thrombin. Two suppliers donated thrombin concentrates for the NIBSC to make into two thrombin preparations (candidates C and D) with 10,000 ampoules each, and approximately 100 units per ampoule. Twenty-five laboratories worldwide participated in the collaborative study to determine the potencies of these two preparations against both the U.S. thrombin standard (Lot J) and the international standard for a-thrombin (89/588). Preliminary results show that candidate D has the potential to unify the two units. Its geometric mean is 110 units per ampoule against both standards using plasma as well as human and bovine fibrinogen as substrate. The result of this study was presented at the 48th Scientific and Standardization Committee meeting of the International Society on Thrombosis and Haemostasis in Boston on 18 July 2002. The report was well received by the committee. Stability studies of the two candidates are now underway and expected to be completed by December 2002. The final report will be sent to all the participants to get approval for choice and unitage of the new standard. Thereafter, the final report, including the responses will be sent to the Expert Committee on Biological Standardization at the World Health Organization in Geneva early in 2003 before their meeting in February when we hope the new international standard will be established. 4. The Correlation of Closure Time and vWF Activities of Factor VIII/vWF Concentrates SUMMARY OF WORK: To explore new test methodology, we investigated the PFA-100 system as a device for detection of vWF activity in factor VIII concentrates. Results: Seven factor VIII concentrates from 7 different manufacturers, and plasma cryoprecipitate were used in this study. VWF activities were tested by ristocetin cofactor (RCof) assay, for collagen binding, and for vWF antigen by ELISA. VWF multimers were detected by the electrophoresis of vWF on high resolution agarose gels (2%), followed by Western-blotting. To determine the relationship of closure time and vWF activity of factor VIII concentrates, a mixture of type III vWD blood (no detectable level of vWF) and factor VIII concentrate was incubated at room temperature for 10 minutes prior to testing on the PFA-100. Each dilution was tested on the PFA machine in duplicate with the collagen/epinephrine and collagen/ADP cartridges. We found that vWF with high molecular weight multimers plays an essential role for platelet anchorage to collagen and platelet aggregation in the PFA-100 system. Factor VIII concentrates with only low molecular weight multimers of vWF did not support the occlusion. There was no good correlation between the closure time and vWF:RCof activity of factor VIII concentrates. One unit of vWF:RCof activity did not always give identical closure time for the different factor VIII concentrates, suggesting that the shear stress assay measures the property of vWF which was not assessed by the RCof test. The relationship of the closure time and clinical outcome is under investigation. 5. Standardization of Recombinant Factor VIII SUMMARY OF WORK: Several studies have shown that recombinant FVIII products often have discrepant potencies when different assay methods are applied. In the case of one recombinant product, use of the chromogenic assay, mandated in Europe, would mean that 30-50% less FVIII would be delivered to patients on a molar basis than if one applied the standard one-stage clotting assay used in the United States. It is important for us to discern the mechanism responsible for such discrepancies, to achieve standardization of potency measurements for recombinant FVIII. Results: Thrombin catalyzes the activation of FVIII by cleaving several peptide bonds located on the heavy and light chains of the FVIII molecule. To evaluate the effect of the acidic regions of FVIII on thrombin-catalyzed activation, we first asked whether there is a complementary binding site(s) on thrombin in addition to the catalytic site for FVIII. gamma-Thrombin, blocked at its active site by D-Phe-Pro-Arg Chloromethyl Ketone (PPACK), inhibited thrombin activation of FVIII. This suggests that a site(s) on thrombin, other than the active site, is needed for FVIII activation. Thrombin-catalyzed cleavages of both the heavy and light chains of FVIII were inhibited by a sulfated polypeptide segment of the FVIII heavy chain, residues 714-740 (F8II). F8II did not inhibit either the amidolytic or the clotting activity of thrombin. This further supports the involvement of sites on thrombin, other than the active site, in FVIII activation. To investigate the potential importance of the fibrinogen-recognition exosite of thrombin on FVIII cleavage, the C-terminal dodecapeptide of hirudin named Hirugen, which binds to the exosite, was tested as an inhibitor of thrombin proteolysis of FVIII. The peptide only partially inhibited the digestion of FVIII, mainly on the light chain, by gamma-thrombin. Furthermore, gamma-thrombin, which lacks the fibrinogen-recognition exosite, cleaved both the heavy and light chains of FVIII, at positions similar to those cleaved by gamma-thrombin. We conclude that a novel recognition site on thrombin, in addition of the fibrinogen-recognition exosite and the active site of thrombin, is involved in activation of FVIII. An acidic region (residues 714-740) of FVIII binds to one of the sites on thrombin involved in the rate-limiting step of the reaction.